Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Since the first report by Gossen and Bujard (Gossen and Bujard, 1992) and subsequent documentation of a variant form (Gossen et al., 1995), the Tetracycline (Tc)-regulated system, has been broadly adopted and is widely acknowledged as the method of choice, in experiments requiring inducible expression of genes of interest. In its originally reported form, the system employs two plasmids. One expressing the tTA or rtTA cDNA (henceforth jointly referred to as TA), a fusion protein of the bacterial Tc-repressor, fused to the C-terminal acidic activation domain of the Herpes Simplex virus (HSV), VP16 transcriptional transactivator. The second plasmid enables cloning of a cDNA of interest downstream of a heptamerized Tc-operator transcription regulatory DNA sequence, fused to a DNA element providing basal promoter activity, derived either from the CMV IE or HSV thymidine kinase promoters Establishing a cell line having Tc-regulatable expression of the gene of interest involves a two step process. In the first, a cell line stably expressing the TA cDNA is established and identified by clonal selection and expression analysis through transient transfection with a Tc-responsive reporter. In the second step, the gene of interest cloned under control of the Tc-responsive element is introduced into the cell line made in the previous step and a second round of selection is performed to identify clones displaying Tc-responsive inducibility of the cDNA (Gossen and Bujard, 1992; Gossen et al., 1995). The Tc-regulated system has effectively overcome several drawbacks seen in earlier systems which showed high basal levels of expression, poor responsiveness and toxicity of the inducing agent. The Tc-inducible system is in addition, able to achieve induction over ranges of several orders of magnitude in a graded manner, responsive to varying levels of inducer. Furthermore, the system is extremely versatile and amenable to several types of modifications, permitting the study of the role of a particular gene, or combinations thereof, in a wide variety of cell types of interest. The potential to use this system in medical applications including gene therapy protocols and pharmacological small molecule screening are areas of active investigation. Its versatility has enabled adaptation to situations requiring inducible gene expression in a tissue specific or generalized manner in animal or plant models, opening new avenues to study gene function in vivo.
The Tc-inducible expression system has been modified in several ways, in attempts to improve performance or tailor it to specific needs. Autoregulatory control was achieved by placing both the tTA as well as exogenous cDNA under control of Tc-operator sequences (Shocket et al., 1995), which reportedly permitted regulation of available tTA levels only on induction and thereby increased overall performance in terms of inducibility and frequency of positive clones obtained. Single plasmid vectors containing the tTA sequence and gene of interest in opposite orientations have been developed to obviate the need for multiple rounds of clonal selection (Baron et al., 1995; Schultze et al., 1996; Weinmann et al., 1994). Overcoming a sometimes considerable barrier of introduction of DNA into transfection recalcitrant cells has been made possible through the development of retroviral vectors for delivery of both components of the system in either a single or combination of two separate viruses (Bohl et al., 1997; Hofmann et al., 1996; Kringstein et al., 1998; Paulus et al., 1996; Rossi et al., 1998). Several promoters have been used to enable generalized or tissue specific expression of tTA in plants (Weinmann et al., 1994) or animals (Efrat et al., 1995; Fishman et al., 1994; Furth et al., 1994; Hennighausen et al., 1995). Modification of the Tc-operator containing plasmid to reduce leaky expression or reduce the effects of integration site has been attempted. Strategies toward this end include Epstein Barr virus (EBV) replication origin based vectors that are maintained episomally (Jost et al., 1997), modified basal promoters to reduce uninduced expression (Hoffmann et al., 1997) and incorporation of sequences that prevent interference from adjoining elements at the site of integration (Hennighausen et al., 1995; McKnight et al., 1992; Stief et al., 1989).
The original report and several other studies have documented potential pitfalls and have provided troubleshooting strategies using the Tc regulated system (reviewed in Blau and Rossi, 1999; Gossen et al., 1994; Shockett and Schatz, 1996)). However, anecdotal evidence non-rigorously documenting failure to establish cell lines that show any significant levels of expression or inducibility of the exogenously introduced gene (Ackland-Berglund and Leib, 1995; Gossen and Bujard, 1995) exists. Drawing upon previous experiences using expression constructs with strong viral promoters based on CMV or SV-40 derived sequences, extinction of expression of transactivator function could be a potentially significant factor encountered in the inability to establish Tc-responsive cell lines. This might be of special relevance in cells having a relatively slow growth rate and/or the potential to differentiate, making them particularly sensitive to this phenomenon, since changes in cell physiology could affect the activity of exogenously introduced viral promoter constructs. The time lapsed between establishing the initial TA expressing clone and identification of cell lines inducibly expressing the gene of interest, is of a sufficient duration, during which the host cell possibly stops supporting CMV promoter enhancer expression, resulting in the shutdown of TA expression. Despite the recent introduction of retroviral vectors that enable single step and therefore relatively quick selection of positive clones, several of these also depend on viral promoters for expression of one or more elements and are therefore also prone to similar problems. The construction of a specific retrovirus is in itself time consuming and a not as yet routine procedure in many laboratories, compared to transfection or electroporation of plasmid DNA into cells. Based on these factors modification of the existing construct for rtTA cDNA expression was done by placing it under the regulation of the human Protein Translation Peptide Elongation Factor-1 xcex1 promoter (EF-1xcex1). This gene has a housekeeping function in all cells and has been documented to be expressed to relatively high levels. More importantly, due to its indispensable housekeeping function in all cells, Protein Translation Peptide Elongation Factor-1 xcex1 promoter (EF-1xcex1) expression is consistent from a temporal viewpoint, relatively insulated from changes in cell physiology and is cell type independent (Goldman et al., 1996; Kim et al., 1990; Wakabayashi-Ito and Nagata, 1994). Utilization of this construct in cells lines derived from diverse human tissues enabled the successful construction of Tc-regulatable lines in every case attempted so far. This modified vector will not only be of general utility but will be especially useful in cases where difficulties have been previously experienced in successfully establishing Tc-responsive clones.
The present invention provides a cell comprising the vector set forth above. The present invention further provides that the cell is from a cell line. The present invention further provides that the cell line is HeLa (human cervix), HO-1 (human melanoma), MCF-7(human breast), PC3 (human prostate) or DU-145 (human prostate).
The Invention also provides an animal comprising the vector set forth above. This invention also provides an animal which comprises a cell which comprises Protein Translation Peptide Elongation Factor-1 xcex1 promoter and nucleic acids encoding reverse tetracycline controlled transactivator, wherein the expression of said transactivator is under the control of Protein Translation Peptide Elongation Factor-1 xcex1 promoter. This invention also provides the animal includes but is not limited to a mouse.
The present invention provides a method of generating a reverse tetracycline controlled transactivator expression system for inducible tetracycline regulated gene expression comprising: (a) isolation of a DNA fragment encoding the reverse tetracycline controlled transactivator by restriction enzyme digestion (b) generation of Protein Translation Peptide Elongation Factor-1 xcex1 promoter vector, by restriction enzyme digestion (c) directional cloning of reverse tetracycline controlled transactivator into Protein Translation Peptide Elongation Factor-1 xcex1 promoter vector by ligation of 5xe2x80x2 EcoRI compatible restriction enzyme overhangs (d) directional cloning of reverse tetracycline controlled transactivator into Protein Translation Peptide Elongation Factor-1 xcex1 promoter vector by Klenow fragment mediated blunt end generation of 3xe2x80x2 Bam HI end of DNA fragment encoding the reverse tetracycline controlled transactivator and 3xe2x80x2 Xbal end of Protein Translation Peptide Elongation Factor-1 xcex1 promoter vector and (e) blunt cloning of partially ligated fragment to produce Protein Translation Peptide Elongation Factor-1 xcex1 promoter vector expressing reverse tetracycline controlled transactivator.
This invention provides the fragment includes but is not limited to an Eco RI-BAM HI fragment, the mammalian expression vector includes but is not limited to pCDEF3, cloning is at the 5xe2x80x2 Eco RI and 3xe2x80x2 BAM HI of the insert and the ligation is at the 5xe2x80x2 Eco RI site and the 3xe2x80x2 Xbal site of pCDEF3.
This invention provides a method of screening pharmacological products using the vector. Finally, this invention provides a method for monitoring inducible gene expression in a tissue specific of generalized manner using the vector.